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研究生:詹凱婷
研究生(外文):Kai-Ting Zhan
論文名稱:蒲公英萃取物對於小鼠巨噬細胞株RAW264.7之抗發炎研究
論文名稱(外文):The anti-inflammatory effect of dandelion extracts on macrophage cell line RAW 264.7
指導教授:邱駿紘邱駿紘引用關係喬長誠喬長誠引用關係
指導教授(外文):Chun-Hung ChiuCharng-Cherng Chyau
學位類別:碩士
校院名稱:弘光科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:178
中文關鍵詞:蒲公英抗發炎
外文關鍵詞:DandelionAnti-inflammatory
相關次數:
  • 被引用被引用:4
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  • 收藏至我的研究室書目清單書目收藏:1
蒲公英具有利膽、利尿及抗風濕的特性,長期以來被認定為傳統的民俗用藥。因其尚有許多健康效益之功效,目前在中草藥市場上還是極為常見。在動物實驗中,蒲公英萃取物被證實有抗發炎的活性,而蒲公英花的區分物亦具有抗氧化,抗血管新生等功效。本研究探討蒲公英水萃取物及酒精萃取物的抗氧化性及其對小鼠巨噬細胞RAW264.7經由脂多醣(LPS)誘導之ㄧ氧化氮、腫瘤壞死因子-���n(TNF-�恁^及介白素(IL-6)等發炎調節物質生成的影響,並闡述其可能調控的相關機轉。
研究結果顯示,在體外抗氧外實驗上,蒲公英水萃取物及酒精萃取物在DPPH自由基清除率及還原力方面具有類似的功效,然而只有水萃取物有螯合亞鐵離子的能力。在總多酚化合物及類黃酮的含量上,分析結果顯示水萃取物高於酒精萃取物,分別為262.2mg;110.0mg及152.5mg;49.5mg。在抗發炎機轉上,不論是水萃取物或酒精萃取物隨著蒲公英萃取物劑量的增加,能降低因脂多醣誘發的一氧化氮產生量。而且,TNF-���n及IL-6的產生量也因蒲公英萃取物的添加而被抑制。與脂多醣單獨處理比較,蒲公英萃取物的存在可使誘導型一氧化氮合成酶(iNOS)的蛋白質及mRNA 表現下降。蒲公英萃取物的添加也會增加I�羠及NF-�羠的表現量。綜合上述之結果蒲公英可以應用在巨噬細胞調控之發炎性疾病的控制,或許可進一步成為改善發炎異常的輔助代替物質。
Dandelion have long been used as the folk medicine for their choleretic, diuretic and antirheumatic properties and are available today in market for a variety of health benefits. The anti-inflammatory activity of dandelion extracts has been confirmed in animal studies and dandelion flower fractions have been shown to possess both antioxidantive and antiangiogenic properties. Expanding upon from current understanding, we examined the effects of aqueous extract of dandelion(AED)and ethanolic extract of dandelion(EED)on lipopolysaccharide(LPS)- induced nitric oxide(NO), tumor necrosis factor-�恁]TNF-�恁^and interleukin-6(IL-6)productions using macrophage RAW264.7 cells and revealed the possible mechanisms underlying.
Both AED and EED displayed similar patterns on DPPH scavenging activity and reducing power, while only AED exhibited ferrous ion chelating ability. The contents of total phenolic compounds and flavonoids were higher in AED as compared with those of EED(262.2 mg and 110.0 mg vs. 152.5 mg and 49.5 mg).
The results indicate that AED and EED inhibited LPS-induced NO production in a dose-dependent manner. Furthermore, AED and EED also decreased the levels of IL-6 and TNF-��. In the presences of AED and EED inducible nitric oxide synthase(iNOS)protein and mRNA levels were declined significantly as compared with LPS induction alone. Both I�羠 and NF-�羠 expressions were also increased with the supplementation of AED and EED. These findings implicate that dandelion can be applied in the regulation of macrophage-mediated inflammatory diseases.
目 錄
目錄…………………………………………………………………………..Ⅰ
圖目錄………………………………………………………………………..Ⅲ
表目錄………………………………………………………………………..Ⅶ
中文摘要………………………………………………………………………1
英文摘要……………………………………………………………….……...2
第一章 緒論………………………………………………………………….3
第二章 文獻回顧…………………………………………………………….5
第一節 蒲公英……………………………………………………………5
第二節 巨噬細胞與免疫反應………………………………………….21
第三節 細胞激素(Cytokines)……………………………………….26
第四節 介白素-6(Interleukin-6, IL-6)……………………………….28
第五節 腫瘤壞死因子-�恁]Tumor necrosis factor, TNF-�恁^…………31
第六節 脂多醣(Lipopolysaccharide, LPS)………………………….32
第七節 一氧化氮(Nitric oxide, NO)及誘導型一氧化氮合成(Inducible nitric oxide synthase, iNOS)…………………….36
第八節 轉錄因子(Nuclear factor-�羠, NF�羠)………………………42
第九節 環氧化酶(Cyclooxygenase, COX)………………………….45
第十節 酚類化合物(Phenolic compounds)…………………………46
第三章 研究目的與實驗架構……………………………………………...51
第四章 材料與方法…...…………………………………………………....53
第一節 實驗試劑與材料……………………………………………….53
第二節 蒲公英萃取物的製備………………………………………….57
第三節 一般成份分析(Proximate Composition Analysis)…………58
第四節 蒲公英萃取物抗氧化能力之評估……………………………60
第五節 細胞培養(Cell culture)……………………………………..66
第六節 細胞毒性測試(MTT assay)…………………………………68
第七節 錐蟲藍染色法(Trypan blue exclusion)…………………….69
第八節 一氧化氮(NO) 濃度分析……………………………………..70
第九節 酵素免疫分析法(ELISA)…………………………………...71
第十節 西方墨點法(Western blotting)……………………………..72
第十一節 反轉錄聚合酶鏈鎖反應(RT-PCR)………………………75
第十二節 統計方法……………………………………………………..78
第五章 實驗結果……………………………………………………………79
第一節 蒲公英之ㄧ般成分分析……………………………………….79
第二節 蒲公英萃取物之收率………………………………………….82
第三節 蒲公英植物化學成分測定…………………………………….83
第四節 蒲公英抗氧化能力分析……………………………………….84
第五節 HPLC-MS分析……………………………………………..….101
第六節 蒲公英萃取物對巨噬細胞RAW264.7細胞存活率之影響…102
第七節 不同濃度之脂多醣對巨噬細胞RAW264.7誘發之細胞毒性及
對一氧化氮生成之影響……………………………………….116
第八節 不同濃度之蒲公英萃取物對脂多醣所誘導之ㄧ氧化氮產生量的影響…………………………………………………………...122
第九節 蒲公英萃取物對脂多醣在不同的誘發時間內,刺激RAW264.7細胞產生一氧化氮、TNF-���n及IL-6的作用………………...131
第十節 蒲公英萃取物對脂多醣所誘導之iNOS、IL-6及TNF-�悀吮RNA表現量的影響………………………………………….144
第十一節 蒲公英萃取物對脂多醣所誘導之抗發炎活性蛋白質的表 現量……………………………………………………..…..149
第六章 討論………………………………………………………………....158
第七章 結論………………………………………………………………....162
參考文獻……………………………………………………………………....163
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